The development and spread of antibiotic resistance in bacteria is a universal threat to both humans and animals. New therapies against multidrug resistant infections are urgently needed. The majority of currently available antibiotics have low efficacy against Gram-negative pathogens because of active efflux of drugs from cells by multidrug efflux transporters. These transporters are promising targets in development of small molecule efflux inhibitors (EPIs) that could be used in combinations with antibiotics to improve their efficacy against Gram-negative pathogens. Our long-term goal is to understand the molecular mechanism of drug efflux in Gram-negative bacteria and to develop approaches to inhibit multidrug efflux transporters. During the previous funding period, using a combination of biochemical, genetic and biophysical approaches we have reconstructed a sequence of events leading to the assembly of active drug efflux complexes and characterized the roles of each component in this process. Our findings exposed a previously unknown vulnerability of multidrug pumps that could be targeted in development of new inhibitors. The objective of this application is to characterize this vulnerability in molecular details and to discover new effectiv inhibitors of drug efflux in Gram-negative bacteria. The central hypothesis is that periplasmic membrane fusion proteins control the transition in efflux pumps from the dormant to the active state, and that inhibition of this transition is an effective way to block multidrug efflux in Gram negative pathogens. The experimental approach is based on molecular analyses of biochemical properties of efflux complexes in the presence of EPIs and the rational design of new inhibitors. We will pursue three specific aims: (i) to investigate the activation of multidrug efflux pumps; (i) to investigate the mechanisms of drug efflux inhibition; (iii) to identify new allosteric inhibitor of drug efflux transporters. The expected outcome of the proposed studies is detailed understanding of how multidrug efflux pumps are activated and new allosteric EPIs acting on this critical step in drug efflux. This contribution is significant because EPIs are expected to restore activities of already existing antibiotics and expand therapeutic options against multidrug resistant infections.